The present invention relates to methods of preparing standard diagnostic gene transcript patterns, probe kits for diagnosis and methods of diagnosis using such kits.
There are numerous examples of diagnostic methods that include physical, anatomical and behavioural examination and/or biochemical, electrical, or electromagnetic studies and/or assays.
These diagnostic methods are well developed and are often efficient means to identify many pathological conditions. They are based on recent developments and research as well as on the observations, experience and empirical data recorded by health-workers concerned with diseases of human beings, other animals and plants for at least 6000 years.
Never before has the arsenal of diagnostic tools been greater than at present, but even so, incorrect diagnosis of ailments and other conditions is still commonplace.
New diseases and conditions are found that may be related to environmental changes or mutations or other alterations in both the active agents or organisms as well as in the organism that is exposed. In addition a number of old and new illegal substances used in sports and by drug addicts do not have appropriate diagnostic tests for their presence.
Several conditions are not easily identified with the available methods and/or the conclusive identification of a disease or condition may be reached too late for adequate corrective treatment.
Due to the extensive time often encountered in a complete diagnostic procedure, incorrect antibiotic therapy is often started prematurely, before a conclusive diagnosis is reached. This medical practice can aggravate the serious development of bacterial strains resistant to antibiotics.
Thus, even though a great number of differential diagnostic methods have been developed, there is still a considerable number of closely related conditions, or combinations of conditions that resist quick, safe and sure identification at low cost.
Furthermore, a number of diagnostic methods depend on the injection of foreign fluids or other kinds of transfer of diagnostic aids onto or into the organism under observation, or require biopsies. The removal of sample tissue from parts of the organisms often not easily accessible may also have a detrimental effect on the identification process and healing process itself.
It is known that certain diseases result in the elevated expression of different genes which may in some cases account for the pathogenicity of the disease or condition in question. Screening for the presence of a particular transcript as an indication of the presence of disease has thus been described (see for example Enderlin et al., FEBS Letters, (1997), 412, p629-632). Methods for quantifying the levels of different transcripts by binding to cDNA derived from gene libraries, or by sequencing and electronic comparison to other libraries has been described in for example Schena et al., (1996), PNAS USA, 93, p10614-10619; Schena et al., (1995), Science, 270, p467-470, Heller et al. (1997), PNAS USA, 94, p2150-2155 and International Patent Application No. WO95/20681.
However, a quick and simple method using the characteristic pattern of gene expression during disease or other conditions or stages thereof as a tool for diagnosis and/or prognosis, particularly a method which does not require any knowledge of the disease characteristics, the genes involved or their sequences has not been described.
It has now surprisingly been found that a simple method of diagnosis of a disease or condition or stage thereof may be performed by the preparation of a characteristic gene transcript pattern standard or fingerprint (standard diagnostic probe pattern) for that disease/condition followed by comparison of the transcript pattern of a patient or organism under study to that pattern. The standard is prepared by the identification of a number of specific and informative probes which serve as a set of markers for the disease or condition or stage thereof to be identified. These probes are bound to a solid support and then hybridized to mRNA, optionally reverse transcribed and/or amplified. The amount of the nucleic acid material which binds to the different probes is assessed and together forms the transcript pattern standard of that disease or condition or stage thereof.
Thus, in order to identify diseases, malaises or other conditions caused by other organisms, toxins, stress, ageing, environmental changes, etc, in human beings, animals, plants and all other living eukaryotic and prokaryotic organisms, a set of standard probe patterns of the amount of transcript from one or more informative genes relative to a standard may be developed, each such standard probe pattern being characteristic of one ailment or condition and/or stage of such ailment. These standard probe patterns are subsequently compared with a pattern of transcript levels, using the same probes, prepared from a recent sample of tissue or body fluid collected from a patient to be diagnosed, such patterns being specific to the present condition of the patient.
Usually the reaction to infections, toxins or deteriorations are accompanied by changes in the level of activity in several or many genes. These activity levels, that may either be relatively higher or lower, are together specific to the type of condition that is encountered. The normal activity and the altered activity may, to a large extent, be measured by the amount of specific transcript or mRNA that is present. Thus, standardised probes for analysis may be designed that have patterns of activity that are characteristic for each condition or combination of conditions that is to be identified or diagnosed. These standardised probes may be used to compare the standardised probe pattern with transcript patterns from samples of tissue or body fluids prepared in a similar way and obtained from a live patient or the organism to be studied.
In order to put the invention into practical use, two kinds of substantially similarly developed diagnostic probes must be available for comparison.
1. A standard diagnostic probe pattern (SDPP) that is characteristic of the suspected ailment, developed from one or more organisms having the condition or disease or stage thereof in question.
and
2. A patient specific probe pattern (PSPP) that is developed from a recently obtained sample of tissue or body fluid from a organism to be studied.
To apply the invention to a specific condition, the pattern of a SDPP characteristic of the suspect ailment or stage thereof must have been developed beforehand. In addition, a recent, well preserved sample of tissue or body fluid from the patient must be available to develop the PSPP, for comparison with one or several different SDPPs for the number of ailments and their different stages that is suspected.
To design and develop the pattern for SDPPs, characteristic for one ailment, known techniques of isolation of mRNA, construction and amplification of cDNA and selection through differential hybridisation and differential display may be used.
Selected informative mRNA or cDNA probes from one or more patients that have been conclusively diagnosed with the ailment in question are isolated and amplified. These SDPPs together will be used to compare if the PSPP is similar to the SDPP. Several such characteristic SDPPs may be developed to represent different stages of the same ailment.
The pattern of such standard probes for a great number of ailments and different stage of such ailments may be accumulated in databases and be made available to laboratories on request.
Thus, viewed from one aspect the present invention provides a method of preparing a gene transcript pattern probe kit for diagnosing or identifying a disease or condition or stage thereof in a prokaryotic or eukaryotic organism comprising at least the steps of:
a) isolating mRNA from the tissue, cells or body fluid of a normal prokaryotic or eukaryotic organism (normal sample);
b) isolating mRNA from the corresponding tissue, cells or body fluid of an organism of step a) having the disease or condition of interest or a stage thereof (diseased sample);
c) separating the mRNA of steps a) and b), which may optionally be reverse transcribed to cDNA, by a non-sequence based separation technique;
d) selecting two or more mRNA or cDNA species which are present at different levels in the normal and diseased samples;
e) isolating the mRNA or cDNA species identified in step d);
f) optionally reverse transcribing the mRNA of step d) or e) to cDNA, unless this has previously been performed in step c); and
g) immobilizing the mRNA or cDNA probes of step e) or f) on one or more solid supports.
As used herein the disease or condition may be any condition, ailment, disease or reaction that leads to the relative increase or decrease in the activity of informative genes of any or all eukaryotic or prokaryotic organisms regardless of whether these changes have been caused by the influence of bacteria, virus, prions, parasites, fungi, radiation, natural or artificial toxins, drugs or allergens, including mental conditions due to stress, neurosis, psychosis or deteriorations due to the ageing of the organism, and conditions or diseases of unknown cause.
Such diseases include those which result in metabolic or physiological changes, such as fever-associated diseases such as influenza or malaria. Other diseases which may be detected include for example yellow fever, sexually transmitted diseases such as gonorrhea, fibromyalgia, candida-related complex, cancer (for example of the stomach, lung, breast, prostate gland, bowel, skin etc), Alzheimer""s disease, disease caused by retroviruses such as HIV, senile dementia, multiple schlerosis and Creutzfeldt-Jakob disease to mention a few.
The invention may also be used to identify patients with psychiatric or psychosomatic diseases such as schizophrenia and eating disorders. Of particular importance is the use of this method to detect diseases, or stages thereof, which are not readily detectable by known diagnostic methods, such as HIV which is generally not detectable using known techniques 1 to 4 months following infection. Conditions which may be identified include for example drug abuse, such as the use of narcotics, alcohol, steroids or performance enhancing drugs. The diagnostic method may be used alone as an alternative to other diagnostic techniques or in addition to such techniques. For example, methods of the invention may be used as an alternative or additive diagnostic measure to diagnosis using imaging techniques such as Magnetic Resonance Imagine (MRI), ultrasound imaging, nuclear imaging or X-ray imaging, for example in the identification and/or diagnosis of tumours.
xe2x80x9cStagesxe2x80x9d thereof refer to different stages of the disease or condition which may or may not exhibit particular physiological or metabolic changes, but does exhibit changes at the genetic level which may be detected as altered gene expression. It will be appreciated that during the course of a disease or condition the expression of different transcripts may vary. Thus at different stages, altered expression may not be exhibited for particular transcripts compared to xe2x80x9cnormalxe2x80x9d samples. However, informative probes are selected from those transcripts which exhibit altered expression at one or more stages through the course of the disease and which together with information relating to the level of other transcripts can be used to provide a characteristic pattern which is indicative of a particular stage of the disease. Such informative probes may be identified by comparing the transcripts of normal samples with transcripts of diseased samples from one or more stages of the disease or condition, or by comparing different stages of the disease or condition to one another, according to the above described method.
As used herein the prokaryotic or eukaryotic organism may be any eukaryotic organisms such as human beings, other mammals and animals, birds, insects, fish and plants, and any prokaryotic organism such as a bacteria.
As used herein the xe2x80x9ctissuexe2x80x9d may be a tissue obtained during surgery, for example by biopsy, or by other means. xe2x80x9cCellsxe2x80x9d include cells isolated from tissues or body fluids or body waste or in the case of prokaryotic organisms, the organism itself. xe2x80x9cBody fluidsxe2x80x9d include urine, blood, semen etc. It will however be appreciated that the method of preparing the standard transcription pattern and method of diagnosis of the invention is also applicable to use on living parts of eukaryotic organisms such as cell lines and organ cultures and explants.
xe2x80x9cNormalxe2x80x9d as used herein refers to organisms or samples which are used for comparative purposes. Preferably, these are xe2x80x9cnormalxe2x80x9d in the sense that they do not exhibit any indication of, or are believed to have, any disease or condition that would affect gene expression, particularly in respect of the disease for which they are to be used as the standard. However, it will be appreciated that different stages of a disease or condition may be compared and in such cases, the xe2x80x9cnormalxe2x80x9d sample corresponds to the earlier stage of the disease or condition. xe2x80x9cDiseasexe2x80x9d samples and organisms include samples and organisms suffering from an ailment or particular condition and are those known to have, or which exhibit, the disease or condition or stage thereof under study.
xe2x80x9cComplementary strandsxe2x80x9d is used in the conventional sense to refer to strands of DNA which are complementary at each base to the template cDNA from which they are derived. xe2x80x9ccDNAxe2x80x9d as referred to herein includes first strand cDNA produced by reverse transcription of RNA and complementary strands to the first strand cDNA, namely second strand cDNA.
xe2x80x9cDifferent levelsxe2x80x9d of the nucleic acids species refers to quantitative or qualitative differences suggestive of differential expression. A xe2x80x9cprobexe2x80x9d may be one or more nucleic acid molecules which may be the same or different (ie. a mixture) but which as a whole are differentially expressed in the normal and diseased samples. When different stages of the disease/condition are investigated, differential expression of the transcripts corresponding to the probes should be exhibited either relative to an undiseased sample or relative to a different stage of the disease/condition. Generally such probes are cDNA reverse transcribed from mRNA, or its complementary strands, although the mRNA itself may also be used. This could for example be achieved using a DNA fragment as a template for the probe by insertion in a vector behind a T3 or similar promoter. It will be appreciated however that this nucleic acid material may be modified without affecting performance of the invention providing hybridization to sample nucleic acid molecules is still possible. Thus the nucleic acid molecules referred to herein, such as mRNA and cDNA, includes molecules which are modified (e.g. methylated) or which include modified or non-natural bases which may be used in the preparation of the cDNA or during amplification. Similar considerations apply to any nucleic acid molecules described herein. Thus for example, the transcripts, present in the samples, may be in the form of RNA or altered to modified forms of RNA and/or into modified or unmodified forms of DNA and/or in the form of, primers, antibodies or other molecules that recognize and bind to target probes, particularly by specific hybridisation to the target probes.
xe2x80x9cAssessingxe2x80x9d as used herein refers to both quantitative and qualitative assessment which may be determined in absolute or relative terms. The characteristic xe2x80x9cpatternxe2x80x9d created by this technique refers to information which for example may be represented in tabular or graphical form and conveys information about the signal associated with two or more probes.
As used herein, reference to xe2x80x9ccorrespondingxe2x80x9d tissues etc. refers to preferably the same tissue, cells or fluid, but also includes tissue, cells or fluid which are sufficiently similar for the purposes of preparing the standard. When used in reference to genes xe2x80x9ccorrespondingxe2x80x9d to the probes, this refers to genes which are related by sequence (which may be complementary) to the probes although the probes may reflect different splicing products of expression. Thus two separate probes may be developed for a single disease which are transcribed from the same gene but reflect different splicing events. However the use of probes which reflect altered gene expression of two or more distinct genes is preferred.
This invention relates to both a diagnostic principle and a method of identifying diseases, malaises and syndromes in any eukaryotic or prokaryotic organism as well as the associated method for the design and development of diagnostic probes to be used in the relative measurements necessary to reach specific diagnosis or to identify relevant conditions.
The invention is a quick and precise method for the diagnosis of any disease or condition that leads to alterations in the activity of genes in a pattern which is specific to any particular condition of the organism under observation.
The ability to design diagnostic standard probes for the identification of traditional conditions, that at present are hard to identify, as well as the ability to quickly adapt the design to prepare new probes for the identification of new conditions that may appear, as soon as they are identified, will therefore be of great value.
From the very early stages of diseases caused by infections, toxic substances, ageing or other conditions changing the quality of life of living eukaryotic organisms, the whole organism responds to the changed condition. The response occurs, throughout the organism, even if only a minor part of the organism appears to be affected. The response lasts until the condition is healed or until the death of the affected organism
The advantages of the invention are of both primary and secondary nature. Samples of tissue or body fluids may be obtained from parts of the organism that are not affected by the condition under observation. One sample will suffice for a complete identification, thus great reductions in costs, time and inconvenience may result by avoiding hospitalization during the normal extensive range of diagnostic tests performed on human and other animal patients.
No foreign substances need be introduced onto or into the organism under observation in order to aid in the identification of the condition, thus the invention will reduce the risk of anaphylactic reactions to such induced diagnostic substances.
The invention has the potential to detect most diseases and syndromes of somatic, psycho-somatic and mental character as well as detecting deterioration due to ageing of the organism. In addition the method may be used to detect the organism""s reactions to toxic substances, radiation, pesticides, antibiotics, drugs, allergens and combinations of several such conditions.
The invention will furthermore make it possible to detect diseases or undesirable conditions in an organism at very early stages, even years before other subjective or objective symptoms may appear.
Even in cases where the patient dies from a hitherto unidentified condition and the cause of death is not established until a forensic post-mortem examination has been performed, the principle will be of value. If, in the attempt to diagnose the patient prior to death, a series of patient specific probe patterns were developed, these probes may be used for the design of new standard probe patterns that may be used to diagnose later occurrences of similar conditions.
The analytical instruments and equipment necessary to make use of the invention is readily available in laboratories engaged in standard biochemical and bio-technological work.
To begin the preparation of the gene transcript pattern probe kit, mRNA is extracted from the tissues, cells or body fluid according to known techniques (see for example Sambrook et. al. (1989), Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) from a normal individual or organism.
mRNA is also extracted, preferably from the same body part of a corresponding individual or organism which has the disease or condition for which the standard diagnostic pattern is to be created. Owing to the difficulties in working with RNA, the RNA is preferably reverse transcribed at this stage to form first strand cDNA, although this may be performed after separation and identification of transcripts of interest if cDNA probes are to be generated. Cloning of the cDNA or selection from, or using, a cDNA library is not however necessary in this or other methods of the invention.
Preferably, the complementary strands of the first strand cDNAs are synthesized, ie. second strand cDNAs, but this will depend on which strands are proposed to be used as the probes and the nature of the nucleic acid molecules in the sample to be probed during the diagnosis method. The second strand cDNA strands are preferably used to probe cDNA strands which have been produced by reverse transcription of the sample mRNA.
Preferably the cDNA strands are amplified by known amplification techniques such as the polymerase chain reaction (PCR) by the use of appropriate primers. Alternatively, the cDNA strands may be cloned with a vector, used to transform a bacteria such as E. coli which may then be grown to multiply the nucleic acid molecules. When the sequence of the cDNAs are not known, primers may be directed to regions of the nucleic acid molecules which have been introduced. Thus for example, as described in the Examples herein, adapters may be ligated to the cDNA molecules and primers directed to these portions for amplification of the cDNA molecules. Alternatively, in the case of eukaryotic samples, advantage may be taken of the polyA tail and cap of the RNA to prepare appropriate primers.
Separation of the normal and diseased sample mRNA or cDNA is performed separately on each sample by non-sequence based separation techniques which are any suitable techniques which allows discrimination between transcripts or their corresponding cDNAs without involving the use of sequence information of particular transcripts to discriminate between the different transcripts/cDNA. This does not however exclude the possibility of for example using probes carrying labels to produce signals on the transcripts if the probes are directed to sequences common on all or most of the transcripts which are not used for the purpose of discriminating transcripts. Thus conveniently, mRNA or cDNA may be separated by electrophoretic separation on an agarose or polyacrylamide gel or a similar gel appropriate for the separation of the nucleic acid molecules. Alternatively, the products may be separated by gas chromatography or HPLC or similar techniques. (Sequence based separation techniques, which are excluded, include for example capture with probes directed to different sequences by hybridization or sequencing itself.)
Such methods offer the advantage that probes to be used in the methods of the invention are identified and selected from the entire population of transcripts or cDNA since no selection is made on the basis of their sequence before separation, e.g. by hybridization to immobilized nucleic acid from control samples. Thus, the identification of the transcripts is not biased towards the selection of particular transcripts from a subset of total transcripts.
To allow comparison between the samples, separation of the transcripts/cDNA should be performed on the normal and diseased samples as simultaneously as possible, e.g. consecutive runs or on the same gel.
To identify the different transcripts or cDNA, it is necessary to identify a signal corresponding to each transcript/cDNA. Conveniently this may be achieved by the use of a radioactive or other label which may be incorporated during cDNA production or during amplification.
Appropriate labels are those which directly or indirectly allow detection or measurement of the presence of the transcripts/cDNA. Such labels include for example radiolabels, chemical labels, for example chromophores or fluorophores (e.g. dyes such as fluorescein and rhodamine), or reagents of high electron density such as ferritin, haemocyanin or colloidal gold. Alternatively, the label may be an enzyme, for example peroxidase or alkaline phosphatase, wherein the presence of the enzyme is visualized by its interaction with a suitable entity, for example a substrate. The label may also form part of a signalling pair wherein the other member of the pair is found on, or in close proximity to, the target probe to which the transcript/cDNA binds, for example, a fluorescent compound and a quench fluorescent substrate may be used. A label may also be provided on a different entity, such as an antibody, which recognizes a peptide moiety attached to the transcripts/cDNA, for example attached to a base used during synthesis or amplification.
A signal may be achieved by the introduction of a label before, during or after the separation step. Thus for example, a gel on which the transcripts have been separated could be probed with labelled polyT oligonucleotides, or cDNA could be probed with labelled polyA oligonucleotides or probed with labelled oligonucleotides directed to a sequence which is introduced by ligation and/or amplification. Alternatively, the presence of transcripts could be identified by other physical properties, such as their absorbance, if techniques such as gas chromatography or HPLC are employed for separation.
Depending on the technique used for separation, signals for different transcripts or their cDNA may overlap and not be fully resolved. Whilst probes may be produced which contain a mixture of transcripts or their cDNA (providing the mix as a whole exhibits differential expression in normal and diseased samples), optionally, the mixture of transcripts/cDNA may be extracted and subjected to repeated or alternative separation techniques to isolate a smaller population of transcripts/cDNAs which exhibits the altered expression.
Nucleic acid species exhibiting differential expression in normal versus diseased samples, or in at least one stage of said disease, are identified. This requires comparison between the signals produced by the normal and diseased samples. Transcripts/cDNAs of interest are those which are differentially expressed in the different samples as exhibited by different amounts of signal. This may correspond to a species present in the diseased sample and not in the normal sample or vice versa. In this way both gene expression which is turned on and gene expression which is turned off is reflected. This offers significant advantages over prior art methods in which altered gene expression is identified relative to normal samples (in which cDNA from normal libraries is used as the hybridization template to which sample mRNA is bound and the relative difference between xe2x80x9cnormalxe2x80x9d and xe2x80x9cdiseasedxe2x80x9d samples are measured). The method of the invention thus can identify genes which are not constitutively expressed or are expressed only at very low levels in normal samples, but are activated in the disease/condition, or at least one stage thereof, in question.
Transcripts/cDNA which reflect a variation in the extent of expression may be used, but it is preferable to use those species which reflect marked differences, e.g. absence or presence of a transcript/cDNA.
Once identified, the mRNA or cDNA species (which may be a mixture of molecules having different sequences) are isolated. To prepare a standard diagnostic pattern, two or more species (probes) which reflect altered gene expression in at least one stage of said disease or condition may be isolated. Whilst in some cases only two probes may be sufficient to produce a standard diagnostic pattern for a particular condition or disease or stage thereof, it will be appreciated that increasing the number of probes will prevent the possibility of misdiagnosis by comparison to other diseases which could similarly alter the expression of the particular genes in question. Thus preferably between 2 and 1000 probe species are selected for isolation, especially preferably between 10 and 500, particularly preferably 50 and 100, for example 70 probe species. These probes reflect genes which have altered expression in the diseases or conditions in question, or particular stages thereof are considered xe2x80x9cinformativexe2x80x9d for that particular disease in the organism under study and only those probes having this informative property are selected.
Isolation may be by selection of appropriate fractions if for example separation was performed on a column, or by physical removal from the separation matrix, for example excision of gel slices containing the nucleic acid species of interest. The nucleic acid molecules contained therein are then isolated and purified if necessary for the subsequent steps, preferably with amplification.
In instances in which the transcripts themselves have been separated and isolated, these may then be converted to cDNA, preferably with amplification.
The mRNA or cDNA probe species produced according to the above method are then each immobilized onto a solid support to produce the disease""s or condition""s probe kit or gene transcript pattern (or fingerprint) probe kit. Numerous solid supports suitable as immobilizing moieties for nucleic acid molecules are well known in the art and widely described in the literature and generally speaking, the solid support may be any of the well-known supports or matrices which are currently widely used or proposed for immobilization, separation etc. in chemical or biochemical procedures. Thus for example, the immobilizing moieties may take the form of particles, sheets, gels, filters, membranes, microfibre strips, tubes or plates, fibres or capillaries, made for example of a polymeric material e.g., agarose, cellulose, alginate, teflon, latex or polystyrene. Particulate materials, e.g. beads, are generally preferred. Conveniently, the immobilizing moiety may comprise magnetic particles, such as superparamagnetic particles.
Attachment of the nucleic acid molecules to the solid support may be performed directly or indirectly. For example if a filter is used, attachment may be performed by UV-induced crosslinking. Alternatively, attachment may be performed indirectly by the use of an attachment moiety carried on the nucleic acid molecules and/or solid support. Thus for example, a pair of affinity binding partners may be used, such as avidin, streptavidin or biotin, DNA or DNA binding protein (e.g. either the lac I repressor protein or the lac operator sequence to which it binds), antibodies (which may be mono- or polyclonal), antibody fragments or the epitopes or haptens of antibodies. In these cases, one partner of the binding pair is attached to (or is inherently part of) the solid support and the other partner is attached to (or is inherently part of) the nucleic acid molecules.
Attachment of appropriate functional groups to the solid support may be performed by methods well known in the art, which include for example, attachment through hydroxyl, carboxyl, aldehyde or amino groups which may be provided by treating the solid support to provide suitable surface coatings. Attachment of appropriate functional groups to the nucleic acid molecules of the invention may be performed by ligation or introduced during synthesis or amplification, for example using primers carrying an appropriate moiety, such as biotin or a particular sequence for capture.
The individual probes form modules of the kit and may be present on one or more solid supports. The solid support of the different modules are conveniently physically associated although the signals of each probe must be separately determinable. Thus for example, plates with multiple wells may be used as the solid support with different probes in the different wells, or regions of a solid support may comprise the different modules, for example the different mRNA or cDNA probes may be bound to a filter at discrete sites.
Thus in a preferred aspect the present invention provides a method of preparing a gene transcript pattern probe kit for diagnosing or identifying a disease or condition or stage thereof in a prokaryotic or eukaryotic organism comprising at least the steps of:
a) isolating mRNA from the tissue, cells or body fluid of a normal prokaryotic or eukaryotic organism (normal sample);
b) isolating mRNA from the corresponding tissue, cells or body fluid of an organism of step a) having the disease or condition of interest or a stage thereof (diseased sample);
c) reverse transcribing the mRNA of steps a) and b) to cDNA;
d) optionally amplifying said strands, optionally incorporating a label into said strands;
e) separating the cDNA of step d) by a non-sequence based separation technique;
f) selecting two or more cDNA species which are present at different levels in the normal and diseased samples;
g) isolating the cDNA species identified in step f); and
h) immobilizing the cDNA probes of step g) on one or more solid supports.
The gene transcript pattern probe kits prepared according to the above method for diagnosing or identifying a particular disease/condition or stage thereof in a particular individual/organism or for preparing a standard diagnostic gene transcript pattern, form further aspects of the invention.
Thus viewed from a further aspect the present invention provides a gene transcript pattern probe kit for diagnosing, identifying or preparing a standard diagnostic gene transcript pattern of a disease or condition or stage thereof in a eukaryotic or prokaryotic organism comprising at least the following:
a) one or more solid supports carrying two or more probe species according to the invention corresponding to transcripts which reflect gene expression of one or more selected genes characteristic in the condition or disease or stage thereof in the organism under investigation.
Optionally the kit may also contain information relating to the signals generated by normal or diseased samples, standardizing materials, e.g. mRNA or cDNA from normal and/or diseased samples for comparative purposes, labels for incorporation into cDNA, adapters for introducing nucleic acid sequences for amplification purposes, primers for amplification and/or appropriate enzymes, buffers and solutions.
The use of such kits to prepare a standard diagnostic gene transcript pattern forms a further aspect of the invention.
In a yet further aspect, the present invention provides a method of preparing a standard diagnostic gene transcript pattern characteristic of a disease or condition or stage thereof in a prokaryotic or eukaryotic organism comprising at least the steps of:
a) isolating mRNA from the tissue, cells or body fluid of said organism having the disease or condition or stage thereof, which may optionally be reverse transcribed to cDNA,
b) hybridizing the mRNA or cDNA of step a) to the mRNA or cDNA probes on a kit according to the invention specific for said disease or condition or stage thereof in an organism corresponding to the organism under investigation; and
c) assessing the amount of mRNA or cDNA hybridizing to each of said probes on said solid support(s) to produce a characteristic pattern reflecting gene expression in the sample with the disease, condition or stage thereof of one or more selected genes corresponding to the probes.
To produce the standard diagnostic gene transcript pattern or fingerprint for a particular disease or condition or stage thereof, the above produced probe kit is used to probe mRNA or cDNA of a diseased sample to give a signal for hybridization to each particular probe species bound to the solid support, ie. the kit modules. A standard control gene transcript pattern may be prepared if desired using mRNA from a normal sample. Thus, total mRNA isolated in the same manner as described above, or its cDNA (depending on the complementarity of the probe species on the probe kit module) of a diseased sample (corresponding to the disease to which the probes are directed) (optionally also a normal sample) is hybridized under appropriate conditions to the probe species on the probe kit modules. When both samples are probed, this may be performed consecutively on the same probe kit modules, by simultaneously hydridizing to the modules of a corresponding probe kit.
To obtain an indication of the number of transcripts/cDNA molecules which become bound to the probe kit modules, the signal produced when the transcripts hybridize is detected (e.g. by detection of double stranded nucleic acid molecules or detection of the number of molecules which become bound, after removing unbound molecules, e.g. by washing). In the latter case, preferably labelled mRNA/cDNA molecules are used as the sample, for example by incorporating radiolabelled bases during reverse transcription, the preparation of complementary cDNA strands or amplification. The amount of signal is then assessed for each probe kit module. The assessment may be quantitative or qualitative and may be based on binding of a single transcript species to each probe, a combination of transcripts, or representative forms of the transcripts such as cDNA or modified nucleic acid molecules as described above. It will be appreciated that quantitative results will provide further information for the transcript fingerprint of the disease which is compiled. This data may be expressed as absolute values or may be determined relative to a particular standard or probe module result. The value of the signal from the normal sample may be subtracted from the signal from the disease sample, where the former is determined, although preferably results from test samples are compared to the unstandardized disease fingerprint which is produced.
Furthermore it will be appreciated that the standard diagnostic gene pattern transcript may be prepared using the results of one or more diseased and/or normal samples to identify probes for the probe kit, and one or more disease samples (and normal samples if used) may be used to perform the hybridization step to obtain the standard diagnostic gene transcript pattern.
The use of such kits and standard diagnostic gene transcript patterns for the purpose of identification or diagnosis of a particular disease or condition or stage thereof in a particular organism forms a further aspect of the invention.
Once a standard diagnostic fingerprint or pattern has been determined for a particular disease or condition using the selected probe species, this information can be used to identify the presence, absence or extent of that disease or condition in a different organism or individual.
Thus viewed from a further aspect the present invention provides a method of diagnosing or identifying a disease or condition or stage thereof in a prokaryotic or eukaryotic organism, comprising the steps of:
a) isolating mRNA from the tissue, cells or body fluid of said organism, which may optionally be reverse transcribed to cDNA;
b) hybridizing the mRNA or cDNA of step a) to a kit of the invention specific for said disease or condition or stage thereof in an organism corresponding to the organism under investigation;
c) assessing the amount of mRNA or cDNA hybridizing to each of said probes on said solid supports to produce a characteristic pattern reflecting gene expression of one or more selected genes corresponding to the probes;
d) comparing said pattern to a standard diagnostic pattern prepared according to the method of the invention using a sample from an organism corresponding to the organism under investigation having said disease or condition or stage thereof under investigation to determine the degree of correlation indicative of the presence of said disease or condition or a stage thereof in the organism under investigation.
The degree of correlation which is required to confirm the presence, absence or extent of a disease or condition necessarily takes into account the range of values which are obtained for normal and diseased samples. Although this can be established by obtaining standard deviations for several representative samples binding to the probes to develop the standard, it will be appreciated that single samples may be sufficient to generate the standard pattern to identify a disease if the test sample exhibits close enough correlation to that standard.
The diagnostic method may be used to identify, quantify or diagnose a disease, condition or ailment or its stage or progression, for example cancer in humans and bovine spongiform encephalopathy in cattle. The methods of the invention may also be used to monitor the condition/state of plants, for example to monitor the effects of pollution, or in the case of prokaryotic organisms, to monitor their state during such processes as fermentation or sewage processing.
Owing to the effects that certain exogenous factors or diseases exert on all parts of body, ie. the effects on gene expression are not isolated to the areas of apparent disease, and thus body parts distant from the site of interest, e.g. a tumour, may be analysed. Thus samples may be obtained for testing in a non-invasive manner, such as for example a body fluid, such as blood. Since samples may originate from different parts of the body which may exhibit some differences in their transcription products, samples used to prepare the standard and test samples should preferably be matched. In the diagnosis methods of the invention three different samples are concerned which could be derived from different sources. These are the samples used to prepare the probes, the disease sample used to prepare the standard diagnosis pattern and the test sample. Preferably all samples are derived from comparable sources, but especially preferably at least the disease sample used to prepare the standard pattern and the test sample are derived from a corresponding source.
The following examples are given by way of illustration only in which the Figures referred to are as follows: